Proc. Nat. Acad. Sci. USA Vol. 72, No. 9, pp. 3491-3495, September 1975
Biophysics
Primary acceptor in bacterial photosynthesis: Obligatory role of ubiquinone in photoactive reaction centers of Rhodopseudomonas spheroides (electron transport/electron paramagnetic resonance/iron)
M. Y. OKAMURA, R. A. ISAACSON, AND G. FEHER Department of Physics, University of California, San Diego, La Jolla, Calif. 92093
Contributed by G. Feher, June 16,1975 ABSTRACT Reaction centers were found to bind two ubiquinones, both of which could be removed by o-phenanthroline and the detergent lauryldimethylamine oxide. One ubiquinone was more easily removed tan the other. The lowtemperature light-induced optical and electron paramagnetic resonance (EPR) changes were eliminated and restored upon removal and readdition of ubiquinone and were quantitatively correlated with the amount of tightly bound ubiquinone. We, therefore, conclude that this ubiquinone plays an obligatory role in the primary photochemistry. The easily removed ubiquinone is thought to be the secondary electron acceptor. The low-temperature charge recombination kinetics, as well as the optical and EPR spectra, were the same for untreated reaction centers and for those reconstituted with ubiquinone. This indicates that extraction and reconstitution were accomplished without altering the conformation of the active site. Reaction centers reconstituted with other quinones also showed restored photochemical activity, although they exhibited changes in their low-temperature recombination kinetics and light-induced (g = 1.8) EPR signals. The observed broad (g = 1.8) EPR signal is interpreted in terms of a magnetically coupled ubiquinone.-Fe2+ acceptor complex. A possible role of iron is to facilitate electron transfer between the primary and secondary ubiquinones.
The primary photochemical act in bacterial photosynthesis involves the light-induced electron transfer from a primary electron donor to a primary electron acceptor in a bacteriochlorophyll-protein complex called the reaction center (RC). The primary electron donor is well characterized and is thought to be a specialized bacteriochlorophyll dimer (15). The primary* electron acceptor, on the other hand, has so far eluded definitive identification. (For a recent detailed review on this subject, see ref. 6). Several pieces of evidence concerning its nature have, however, accumulated during the past several years. The presence of stoichiometric amounts of iron in RC's (7) and the observation of a broad electron paramagnetic resonance (EPR) signal (7-9) led to the hypothesis that iron was the primary acceptor. This hypothesis was questioned by Loach and Hall, who reported full photochemical activity in iron-depleted preparations (10). They observed a new, narrow, light-induced EPR signal that was shown to be due to a ubiquinone (UQ) radical (11, 12). In addition, optical absorbance changes in illuminated RC's, similar to those observed in UQ anion radicals, were reported (13-15). To exAbbreviations: RC, reaction center; UQ, ubiquinone; LDAO, lauryldimethylamine oxide; EPR, electron paramagnetic resonance. * By "primary" we mean the first intermediate that can be stabi-
lized for times of the order of milliseconds. We ignore in the present discussion the possibility of transient intermediates, such as bacteriochlorophyll- or triplets, which may have shorter lifetimes.
plain these results, three possible candidates for the primary electron acceptor have been proposed (6): (1) Fe, (2) UQ, and (3) Fe-UQ complex. Recent studies utilizing magnetic resonance and Mossbauer spectroscopy have shown that iron alone cannot be the primary acceptor. RC's in which Fe was replaced by Mn exhibited a different light-induced EPR signal. However, the donor-acceptor (i.e., electron-hole) recombination time remained unchanged (16). Since this recombination time is believed to be a sensitive indicator of the electronic structure and conformational state of the donor-acceptor pair (17), it was concluded that the transition metal ion can play only a secondary role in the primary photoact (16). This was confirmed by Mossbauer studies which showed that the valence of iron was the same (Fe2+) in both the oxidized and reduced acceptor molecule (16, 18). Chemical evidence supporting the role of UQ has come from the experiments of Cogdell et al. (19), who extracted (and re-added) UQ from chromatophores and RC's and observed an accompanying loss (gain) of photochemical activity. However, these experiments involved extractions with organic solvents, which are known to cause serious denaturation of RC's. Consequently, an accurate correlation between photochemical activity and UQ content, as well as the conformation of the reconstituted RC's, was difficult to determine. In the present study we have developed a method for UQ removal and readdition that avoids the use of organic solvents. We have shown that 1.0 UQ per RC is required for the photochemical activity. In order to test whether RC's reconstituted with UQ were in their native conformation, EPR spectra and low-temperature donor-acceptor recombination times were measured. The structural requirements for the reconstitution of active RC's were explored by replacing UQ with other quinones. The main features of the broad (g = 1.8) EPR signal due to the acceptor are accounted for by a
simple model. MATERIALS AND METHODS Preparation of Reaction Centers. RC's were prepared from Rhodopseudomonas spheroides R-26 as previously described (20), except that a lower detergent concentration [1% laurydimethylamine oxide (LDAO) for chromatophores having A8651 cm = 100] was used during the solubilization process. The concentration of the RC's was determined optically using the extinction coefficient e8w2M = 2.88 X 105 M-1
cm-1 (21). Removal of Ubiquinone. Varying amounts of UQ were removed by incubating RC's for 3-6 hr at 250C in the pres3491
Biophysics: Okamura et al.
3492
-
LLJ1.0
_ A800=
c_
Proc. Nat. Acad. Sci. USA 72 (1975)
2,
10 M o-phen
t0.5-
A800y
0.2, 102 M
0-
phen
00 0
2 3 --LDAO CONCENTRATION ['i% W/V]
4
FIG. 1. The removal of ubiquinone from reaction centers of R. spheroides R-26 with LDAO and o-phenanthroline (o-phen). One ubiquinone is weakly bound and can be removed by a mild treatment (upper two curves, incubation time 3 hr), the other is more tightly bound (lower curve, incubation time 6 hr). A second incubation in 4% LDAO, 10-2 M o-phenanthroline, A8W1 cm = 0.2, resulted in less than 0.05 UQ/RC. The binding of ubiquinone to these reaction centers is shown in Fig. 2.
of different amounts of LDAO (1-4%) and o-phenanthroline (1-10 mM). All buffers were 10 mM Tris-HCl, pH 8.0. The RC's were then adsorbed onto a column of DEAEcellulose (Whatman DE 52), washed with 0.1% LDAO, eluted with 1 M NaCl (0.1% LDAO), and dialyzed against 0.025% LDAO. A large-scale preparative procedure for maximal removal of UQ was to adsorb RC's onto a column of DEAE-cellulose that was washed for 10 hr at 250C with 4% LDAO, 10 mM o-phenanthroline (1 liter/30 mg of RC's). The residual UQ in the RC's was extracted for 30 min at 350C in acetone-methanol (1:1, v:v), then purified, following the procedure of K. and A. Takamiya (22), and assayed by measuring the change in absorbance at 275 nm after reduction with NaBH4 [Ae275M = 12.25 X 103 Mcm'1] (23). Reconstitution of Reaction Centers. Reconstitution of UQ-depleted RC's was accomplished by two different methods. 14C-Labeled UQ-50 (Qlo) (about 5 X 10-5 M), solubilized in 1% LDAO, was added to RC's (As'l cm = 2, 1% LDAO) and incubated at 4°C for 10 hr. The RC's were separated from the unbound UQ by DEAE chromatography, as before, and their UQ content was assayed by liquid scintillation counting. An alternate method was used for other quinones that were expected to bind more weakly. Different ratios of UQ/RC (1, 5, 20), solubilized in 1% LDAO, were added to RC's (A 8'l cm = 20, 1% LDAO), incubated first at 25°C for 1 hr and then at 40C for 10 hr. EPR and optical samples were prepared by adding an equal volume of glycerol to the reconstituted RC's and freezing them in liquid nitrogen. This method was tested also with UQ-50j and found to give the same results as the first method. The iron content was measured by atomic absorption spectroscopy (Varian-Techtron model AA-5). Source of Quinones. The radiolabeled UQ was prepared by growing R. spheroides R-26 in the presence of [14C]acetate (0.3 mCi/liter). The [14C]UQ was extracted as described
ence
above and purified by thin-layer chromatography on silica gel sheets (Eastman), developed with benzene, eluted with ethanol, and crystallized from ethanol. The specific activity was 0.1 ;tCi/gnmol. This quinone has been previously shown to be UQ-50 (16). UQ-50, UQ-30, menadione, and phylloquinone were obtained from Sigma Chemical Co.; duroquinone and anthraquinone from Aldrich Chemical Co.; and plastoquinone-C from W. Butler (University of California at San Diego). Determination of Photochemical Activity. The photochemical activity at cryogenic temperatures was determined by two methods: (1) Optically by using the IR-2 mode of the Cary 14 as the strong (actinic) light source and measuring the bleaching at 890 nm. The sample was in thermal contact with a copper "cold finger" that was immersed in liquid nitrogen, and was in a Pyrex dewar with optically flat windows. (2) By measuring the amplitude of the light-induced EPR signal at g = 2.0026. The percent of activity agreed for both cases with the amplitude extrapolated to infinite light intensity obtained from a plot of reciprocal amplitude versus reciprocal light intensity. The kinetics of the light-induced changes were measured as described earlier (17). The EPR spectrum of the broad signal was obtained with light modulation (7, 17) (rather than magnetic field modulation). In this mode of operation the output signal is proportional to the absorption X" rather than the usually observed derivative
dX"/dH. EXPERIMENTAL RESULTS Removal of ubiquinone The UQ content of purified RC's was found to be close to 2 molecules/RC. UQ was removed by incubating RC's in the presence of LDAO and o-phenanthroline. By varying the concentration of protein, LDAO, o-phenanthroline, and the incubation period, different amounts of bound UQ were removed. The number of UQ's that remained bound to RC's under different conditions is shown in Fig. 1. It is seen that one UQ molecule is easily removed (Fig. 1, top two curves) while the removal of the second molecule requires more rigorous conditions (Fig. 1, bottom curve). RC's extracted twice under the most severe conditions (4% LDAO, 10-2 M ophenanthroline, A800' cm = 0.2) were labeled UQ-depleted; they contained about 0.05 UQ/RC and 1.05 i 0.05 Fe/RC. Reducing conditions were found to facilitate the removal of UQ; RC's incubated in the presence of 5 mM NaBH4 (0.5% LDAO, 50 mM Tris, pH 8) were found to lose all UQ in excess of 1.0 UQ/RC. Reconstitution of reaction centers with ubiquinone UQ-depleted RC's were reconstituted with various amounts of UQ. In order to improve the accuracy and ease of determination of UQ incorporation, radiolabeled ['4C]UQ was used. Fig. 2 shows the amount of UQ bound as a function of UQ added. The photochemical activity of untreated, UQ-depleted, and reconstituted RC's was measured at cryogenic temperatures. Two independent criteria were used for measuring the activity: (1) The light-induced change in absorbance of the 890 nm peak (Fig. 3a-c) and (2) the amplitudes of the two light-induced EPR signals (at g = 2.0026 and g = 1.8) corresponding to the donor (D+) and acceptor (A-) (Fig. 3d-f). In UQ-depleted RC's, the light-induced optical and EPR signals were virtually eliminated. However, in the absence of UQ a triplet EPR signal, as reported by Dutton, et al. (25), was observed (see insert in Fig. 3e). Upon addition of UQ to
Biophysics: Okamura et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
this low (2.1OK) temperature, the measurements were performed under partial microwave saturation of the narrow EPR line (16, 17). The quantitative relation between low-temperature photochemical activity and UQ content was determined on two sets of samples. In one, UQ was removed to varying degrees and in the other different amounts of UQ were added to UQ-depleted RC's. The results on both sets of samples are shown in Fig. 4. The light-induced optical changes and EPR amplitudes were normalized to untreated RC's. The dotted line, which approximates the experimental points fairly well, represents the expected light-induced changes if one UQ per RC is required for activity.
2.0
O05 1.0
3493
3
00
Reconstitution of reaction centers with other quinones In order to understand the structural requirements of the primary acceptor, other quinones were used to reconstitute UQ-depleted RC's. A list of quinones that were successfully incorporated is shown in Table 1. No detailed studies of incorporation, like those shown in Fig. 2, were performed; in most cases, however, it was found that a 5-times excess of added quinone resulted in close to maximum low-temperature activity. Two parameters that are believed to reflect the environment of the acceptor were measured: (1) The low-temperature decay time -TD (see previous section), and (2) the line width AH (full width at half amplitude) of the broad acceptor signal at g = 1.8. Both of these parameters changed when RC's were reconstituted with quinones other than UQ and are summarized in Table 1. In several cases, multiphasic decay kinetics were observed; the values of rD listed in Table 1 represent the initial decay.
-UQ ADDED [MOLE FRACTION]
FIG. 2. The binding of ubiquinone (UQ-50) to UQ-depleted reaction centers of R. spheroides R-26 (see Fig. 1). Reaction centers (A'ool cm = 2) in 1% LDAO were incubated with 14C-labeled ubiquinone for 10 hr at 41C.
the UQ-depleted RC's, both the light-induced optical and EPR changes were fully restored (compare Fig. 3a with Sc, and 3d with Sf). The low-temperature decay constant TD, obtained from the time dependence of the EPR amplitude, defined by A(t) = Ao e1t/TD was measured for untreated and reconstituted RC's. At 800K, TiD of the narrow EPR signal was found to be 28 4 2 msec for the untreated and 29 i 2 msec for the reconstituted RC's. The same values of TTD were obtained at 2.10K for both the broad (g = 1.8) and the narrow (g = 2.0026) EPR signals. In order to prevent the electron spinlattice relaxation from becoming the rate-limiting process at 0.8 b :zi
L'i
C-) .Z -cl m
0.6
CD U')
(0.4-
m -el
-i -CZ
(0.2
.2 Q-
C)
f 700
UNTREATED REACTION CENTERS d
750
900 800 850 -'WAVELENGTH [nml
950
UBIQUINONE ADDED BACK
UBIQUINONE REMOVED
T 2.I'K =
e-904 GHz
x'
-j
cr
t g2.0026 0
2
3
g=I.8 4
5
6
7
8
0
2
3
4
5
6
7
8
2
3
4
5
6
7
8
-'-MAGNETIC FIELD (kGoussJ FIG. 3. Low-temperature light-induced optical changes (upper) and EPR signals (lower) of reaction centers of R. spheroides R-26 with and without ubiquinone (UQ-50). The optical bleaching was accomplished by using the Cary in the IR-2 mode. The EPR signals were obtained with 4 Hz light modulation and are, therefore, proportional to the absorption x' and not to the usually observed derivative d x/dH. Microwave power 10-4 W, cavity Q0o 104. All samples were in 50% glycerol solution (optical path length of 1 mm, volume 0.1 ml). EPR samples had A800' cm = 15. Untreated reaction centers (d) and those to which ubiquinone was added back after extraction (f) show a narrow g = 2.0026 signal due to the donor (D+) and a broad g = 1.8 signal due to the acceptor (A-). Reaction centers from which virtually all the ubiquinone had been extracted show negligible bleaching (b) and a small EPR signal (e) attributed to the bacteriochlorophyll (BChl) triplet. The insert in (e) was obtained by increasing the light modulation frequency to 40 Hz (thereby eliminating the residual D+ signal), expanding the magnetic field scale and increasing the gain 5-fold.
Biophysics: Okamura et al.
3494
Proc. Nat. Acad. Sci. USA 72 (1975) Table 1. Low-temperature decay kinetics and EPR line widths of the acceptor in reaction centers of R. spheroides R-26 reconstituted with different quinones*
OPTICAL BLEACHING AA/A AT 890 nm + EPR AMPLITUDE (g92.0026) -____ EXPECTED IF ONE UQ/RC IS REQUIRED FOR ACTIVITY o
T = 80'K "
=
z
100
+>---o+0
4
+
*+
.,
80
UBIQUINONE-50
\0
8 ~\
_
60
LINE WIDTH
29±2
600±30
29±2
600± 30
32±2
680 ± 40
105 ±10
1000±60
37±3
740±40
34±2
760±40
135+10
740±40
28±2
600±30
_
X
+
DECAY TIME
Tt G~s tD msed]~ AH (GAUSS]*
STRUCTURE
COMPOUND
0+
y3 CH30
cn -:I M
40
C2:
20
C=
2
UBIQUINONE-30
2.
.0
b
PLASTOOUINONE-C
1.0 0.5 1.5 UO/RC (REMAINING AFTER EXTRACTION) o + ____
~36
OPTICAL BLEACHING AA/A AT 890nm EPR AMPLITUDE (9-2.0026) EXPECTED IF ONE UO/RC IS REQUIRED FOR ACTIVITY _
-o_
_
DUROQUINONE
CH3wCH3
MENADIONE
XCH3
0
T 80'K _+
CH3X
PHYLLOQUINONE
H
16 33
(VITAMIN K )
_
/
9
O/
ANTHRAQUINONE
T
IO
60
1.40
+,
UNTREATED REACTION CENTERS
2C
0
1.0 1.5 0.5 -UQ /RC (BOUND AFTER RECONSTITUTION)
2.0
FIG. 4. The relation between low-temperature photoactivity (normalized to untreated reaction centers) and the number of ubiquinones per reaction center. (a) obtained with reaction centers exposed to varying concentrations of LDAO and o-phenanthroline; (b) [14C]ubiquinone was added in varying amounts to reaction centers that had less than 0.05 ubiquinone per reaction center. The iron content (about 1 Fe/RC) remained unaffected by the extraction procedure.
DISCUSSION AND CONCLUSION We have shown that RC's bind two molecules of UQ which are distinguishable by their ease of removal with LDAO and o-phenanthroline (Fig. 1). The more loosely bound UQ is not required for the primary photochemistry and probably represents the secondary electron acceptor (6, 26). The ease of displacement of this UQ by o-phenanthroline offers a simple explanation of the inhibitory effect of o-phenanthroline on electron transfer to secondary electron acceptors (27). A possible mechanism of the removal of the UQ's by o-phenanthroline is the latter's binding to Fe2+, thereby displacing the UQ's. This suggests that both UQs' are situated in close proximity of the iron. The observation that reducing conditions facilitate the removal of one UQ is consistent with a mechanism of electron transfer in intact membranes that involves the release of the reduced secondary UQ from the RC to a UQ pool. The tightly bound UQ was found to be quantitatively correlated with the disappearance and recovery of the lowtemperature photochemical activity (Figs. 3 and 4). We conclude, therefore, that in R. spheroides it plays an obligatory role in the primary photochemistry. Several authors working with Rhodospirillum rubrum and Chromatium have concluded that UQ cannot be important in the primary photochemical act (28-30). These conclusions were based on the
* UQ-depleted reaction centers were incubated for 10 hr at 4'C in the presence of different quinones. For details, see Materials and
Methods.
t TD is the time required for the narrow EPR signal (g = 2.0026) to reach l/e of its amplitude after cessation of illumination (measured at T = 80WK). t AH is the full width at half amplitude of the broad g = 1.8 signal (see Fig. 3f), measured at 2.1'K.
presence of the primary reaction after extraction of UQ. It seems to us unlikely that different bacterial species should have developed totally different acceptorst, although this possibility cannot presently be excluded. Two possible alternative explanations for these conflicting results are that either the extraction conditions were not adequate to remove the tightly bound UQ, or that the photochemical activity at room temperature was mediated by an exogenous (diffusible) acceptor. The latter case points out the advantage of using low-temperature kinetic data as a criterion for "native" photochemical activity. The low-temperature decay time rD of the EPR signals, as well as the line width AH of the broad acceptor signal (g = 1.8), were found within experimental error to be the same for untreated and reconstituted RC's. Since these parameters are believed to be sensitive to the detailed environment of the acceptor (e.g., donor-acceptor distance), we conclude that the extraction of UQ and the reconstitution of the RC's were accomplished without altering the conformation of the active site. RC's reconstituted with other quinones showed deviations in the values of TiD and AH (Table 1). A tunneling mechanism for the decay, characterized by TiD, had been previously postulated (17). Consequently, TD should depend in a sensitive way on the shape of the potential barrier and the ionization (redox) potentials of the primary reactants (17). It will be instructive to test the tunneling hypothesis by makt
However, UQ.
as
shown in Table 1, different quinones might replace
Biophysics: Okamura et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
ing a detailed comparison of the observed TrD's and the respective redox potentials with the recently developed tunneling theory of Hopfieldt (31). Irrespective, however, of the detailed mechanism of charge recombination, the fact that replacing UQ with other quinones changes TD, whereas replacing iron by manganese does not, provides an additional argument in favor of assigning to UQ the major role in the primary photoact. It is noteworthy that in spite of the changes in TD and AH, photochemical activity was restored in RC's reconstituted with quinones of different structures. Although the structural requirements for reconstitution have not yet been fully explored, the present results indicate that neither the isoprenoid chain nor the methoxy groups of UQ are absolutely required for binding or photochemical activity. The observation of multiphasic kinetics indicates that in some of the reconstituted RC's the quinone did not assume a unique position. Having established that UQ plays an obligatory role in the primary photochemistry, what then is the function of iron? The chemical evidence [i.e., iron removal (10) and its replacement with manganese (16), the unchanged valence of iron upon reduction of the acceptor (18)] indicates that iron plays a relatively minor role in the primary photoact. On the other hand, spectroscopic evidence (i.e., the appearance of the g = 1.8 EPR signal) indicates that the presence of iron has a large influence on the EPR spectrum of the primary acceptor. These two sets of observations can be reconciled by postulating an Fe-UQ (ferroquinone) complex (11, 12). In the reduced form of the complex, the extra (unpaired) electron is mainly localized on the UQ; it can, however, interact magnetically with the nearby Fe2+. This interaction shifts and broadens (by two orders of magnitude) the quinone EPR resonance line. Preliminary calculations (H. Shore, personal communication) have shown that a magnetic exchange interaction (of the form JSI-S2), between a UQ radical (S, = %) and Fe2+ (S2 = 2) can give rise to g-values similar to those observed§. Since the energies involved in chemical reaction are much larger than magnetic energies, the fact that the EPR spectrum of the acceptor is greatly altered by the presence of iron does not mean that iron is essential for the primary photochemistry. Indeed, the indication that both UQ's are in close proximity to the iron leads to the hypothesis that the function of Fe is to serve as a path for electron transfer between the primary and secondary UQ's. We thank H. Shore for illuminating discussions and calculations on the magnetic properties of the iron-ubiquinone complex, L. Ackerson and S. Giovannoni for technical assistance, and W. Butler for the sample of plastoquinone. This work was supported by National Science Foundation Grant BMS-74-21413 and National Insti-
The
simplified tunneling
model of ref.
17
predicts
a
fourfold
change in 7rD for a 10% (about 0.1 eV) change in the redox potential of the acceptor. § EPR experiments model iron-quinone complexes such a~sFebenzoquinone have been performed by Blumberg and Peisach (32). on
3495
tutes of Health Grant USPHS GM-13191, and by a Career Development Award to M.Y.O. (1 K04 GM00106-01). 1. Norris, J. R., Uphaus, R. A., Crespi, H. L. & Katz, J. J. (1971) Proc. Nat. Acad. Sci. USA 68,625-629. 2. Feher, G., Hoff, A. J., Isaacson, R. A. & McElroy, J. D. (1973) Biophys. Soc. Abstr. 13, 61a (abstr. no. WPM-H7). 3. Norris, J. R., Druyan, M. E. & Katz, J. J. (1973) J. Am. Chem. Soc. 95,1680-1684. 4. Feher, G., Hoff, A. J., Isaacson, R. A. & Ackerson, L. C. (1975) Ann. N.Y. Acad. Sci. 244,239-259. 5. Norris, J. R., Sheer, H. & Katz, J. J. (1975) Ann. N.Y. Acad. Sci. 244, 261-280. 6. Parson, W. W. & Cogdell, R. J. (1975) Biochim. Biophys. Acta 416, 105-149. 7. Feher, G. (1971) Photochem. Photobiol. 14,373-88. 8. McElroy, J. D., Feher, G. & Mauzerall, D. (1970) Biophys. Soc. Abstr. 10, 204a. (abstr. no. FAM-E7) 9. Dutton, P. L., Leigh, J. S. & Reed, D. W. (1973) Biochim. Biophys. Acta 292, 654-664. 10. Loach, P. A. & Hall, R. L. (1972) Proc. Nat. Acad. Sci. USA 69,786-790. 11. Feher, G., Okamura, M. Y. & McElroy, J. D. (1972) Biochim. Biophys. Acta 267,222-226. 12. Bolton, J. R. & Cost, K. (1973) Photochem. Photobhol. 18, 417-421. 13. Clayton, R. K. & Straley, S. C. (1972) Biophys. J. 12, 12211234. 14. Slooten, L. (1972) Biochim. Biophys. Acta 275,208-218. 15. Bensasson, R. & Land, E. J. (1973) Biochim. Biophys. Acta 325, 175-181. 16. Feher, G., Isaacson, R. A., McElroy, J. D., Ackerson, L. C. & Okamura, M. Y. (1974) Biochim. Blophys. Acta 368, 135-139. 17. McElroy, J. D., Feher, G. & Mauzerall, D. C. (1974) Biochim. Blophys. Acta 333,261-278. 18. Debrunner, P. G., Schultz, C. E., Feher, G. & Okamura, M. Y. (1975) Biophys. J. 15, 226a (Abstract no. Th-PM-L12). 19. Cogdell, R. J. Brune, D. C. & Clayton, R. K. (1974) FEBS Lett. 45,344-347. 20. Okamura, M. Y., Steiner, L. A. & Feher, G. (1974) Biochemistry 13, 1394-1403. 21. Straley, S. C., Parson, W. W., Mauzerall, D. & Clayton, R. K. (1973) Blochim. Biophys. Acta 305,597-609. 22. Takamiya, K. & Takamiya, A. (1969) Plant Cell Physiol. 10,
363-373. 23. Pumphrey, A. M. & Redfearn, E. R. (1960) Biochem. J. 76, 61-64. 24. Feher, G., Isaacson, R. A. & McElroy, J. D. (1969) Rev. Sci. Instrum. 40, 1640-1641. 25. Dutton, P. L., Leigh, J. S. & Seibert, M. (1972) Biochem. Biophys. Res. Commun. 46,406-413. 26. Halsey, Y. D. & Parson, W. W. (1974) Biochim. Biophys. Acta
347,404-416. 27. Parson, W. W. & Case, G. D. (1970) Biochim. Biophys. Acta
205,232-245. 28. Noel, H., Van der Rest, M. & Gingras, G. (1972) Biochim. Biophys. Acta 275,219-230. 29. Ke, B., Garcia, A. F. & Vernon, L. P. (1973) Biochim. Biophys. Acta 292,226-236. 30. Bearden, A., Malkin, R., Parson, W. W. & Capolell, R. J., quoted on p. 138 of ref. 6. 31. Hopfield, J. J. (1974) Proc. Nat. Acad. Sci. USA 71, 36403644. 32. Blumberg, W. E. & Peisach, J. (1965) in Non-Heme Iron Proteins, ed. San Pietro, A. (The Antioch Press, Yellow Springs, Ohio), p. 101.
Biophysics
Primary acceptor in bacterial photosynthesis: Obligatory role of ubiquinone in photoactive reaction centers of Rhodopseudomonas spheroides (electron transport/electron paramagnetic resonance/iron)
M. Y. OKAMURA, R. A. ISAACSON, AND G. FEHER Department of Physics, University of California, San Diego, La Jolla, Calif. 92093
Contributed by G. Feher, June 16,1975 ABSTRACT Reaction centers were found to bind two ubiquinones, both of which could be removed by o-phenanthroline and the detergent lauryldimethylamine oxide. One ubiquinone was more easily removed tan the other. The lowtemperature light-induced optical and electron paramagnetic resonance (EPR) changes were eliminated and restored upon removal and readdition of ubiquinone and were quantitatively correlated with the amount of tightly bound ubiquinone. We, therefore, conclude that this ubiquinone plays an obligatory role in the primary photochemistry. The easily removed ubiquinone is thought to be the secondary electron acceptor. The low-temperature charge recombination kinetics, as well as the optical and EPR spectra, were the same for untreated reaction centers and for those reconstituted with ubiquinone. This indicates that extraction and reconstitution were accomplished without altering the conformation of the active site. Reaction centers reconstituted with other quinones also showed restored photochemical activity, although they exhibited changes in their low-temperature recombination kinetics and light-induced (g = 1.8) EPR signals. The observed broad (g = 1.8) EPR signal is interpreted in terms of a magnetically coupled ubiquinone.-Fe2+ acceptor complex. A possible role of iron is to facilitate electron transfer between the primary and secondary ubiquinones.
The primary photochemical act in bacterial photosynthesis involves the light-induced electron transfer from a primary electron donor to a primary electron acceptor in a bacteriochlorophyll-protein complex called the reaction center (RC). The primary electron donor is well characterized and is thought to be a specialized bacteriochlorophyll dimer (15). The primary* electron acceptor, on the other hand, has so far eluded definitive identification. (For a recent detailed review on this subject, see ref. 6). Several pieces of evidence concerning its nature have, however, accumulated during the past several years. The presence of stoichiometric amounts of iron in RC's (7) and the observation of a broad electron paramagnetic resonance (EPR) signal (7-9) led to the hypothesis that iron was the primary acceptor. This hypothesis was questioned by Loach and Hall, who reported full photochemical activity in iron-depleted preparations (10). They observed a new, narrow, light-induced EPR signal that was shown to be due to a ubiquinone (UQ) radical (11, 12). In addition, optical absorbance changes in illuminated RC's, similar to those observed in UQ anion radicals, were reported (13-15). To exAbbreviations: RC, reaction center; UQ, ubiquinone; LDAO, lauryldimethylamine oxide; EPR, electron paramagnetic resonance. * By "primary" we mean the first intermediate that can be stabi-
lized for times of the order of milliseconds. We ignore in the present discussion the possibility of transient intermediates, such as bacteriochlorophyll- or triplets, which may have shorter lifetimes.
plain these results, three possible candidates for the primary electron acceptor have been proposed (6): (1) Fe, (2) UQ, and (3) Fe-UQ complex. Recent studies utilizing magnetic resonance and Mossbauer spectroscopy have shown that iron alone cannot be the primary acceptor. RC's in which Fe was replaced by Mn exhibited a different light-induced EPR signal. However, the donor-acceptor (i.e., electron-hole) recombination time remained unchanged (16). Since this recombination time is believed to be a sensitive indicator of the electronic structure and conformational state of the donor-acceptor pair (17), it was concluded that the transition metal ion can play only a secondary role in the primary photoact (16). This was confirmed by Mossbauer studies which showed that the valence of iron was the same (Fe2+) in both the oxidized and reduced acceptor molecule (16, 18). Chemical evidence supporting the role of UQ has come from the experiments of Cogdell et al. (19), who extracted (and re-added) UQ from chromatophores and RC's and observed an accompanying loss (gain) of photochemical activity. However, these experiments involved extractions with organic solvents, which are known to cause serious denaturation of RC's. Consequently, an accurate correlation between photochemical activity and UQ content, as well as the conformation of the reconstituted RC's, was difficult to determine. In the present study we have developed a method for UQ removal and readdition that avoids the use of organic solvents. We have shown that 1.0 UQ per RC is required for the photochemical activity. In order to test whether RC's reconstituted with UQ were in their native conformation, EPR spectra and low-temperature donor-acceptor recombination times were measured. The structural requirements for the reconstitution of active RC's were explored by replacing UQ with other quinones. The main features of the broad (g = 1.8) EPR signal due to the acceptor are accounted for by a
simple model. MATERIALS AND METHODS Preparation of Reaction Centers. RC's were prepared from Rhodopseudomonas spheroides R-26 as previously described (20), except that a lower detergent concentration [1% laurydimethylamine oxide (LDAO) for chromatophores having A8651 cm = 100] was used during the solubilization process. The concentration of the RC's was determined optically using the extinction coefficient e8w2M = 2.88 X 105 M-1
cm-1 (21). Removal of Ubiquinone. Varying amounts of UQ were removed by incubating RC's for 3-6 hr at 250C in the pres3491
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3492
-
LLJ1.0
_ A800=
c_
Proc. Nat. Acad. Sci. USA 72 (1975)
2,
10 M o-phen
t0.5-
A800y
0.2, 102 M
0-
phen
00 0
2 3 --LDAO CONCENTRATION ['i% W/V]
4
FIG. 1. The removal of ubiquinone from reaction centers of R. spheroides R-26 with LDAO and o-phenanthroline (o-phen). One ubiquinone is weakly bound and can be removed by a mild treatment (upper two curves, incubation time 3 hr), the other is more tightly bound (lower curve, incubation time 6 hr). A second incubation in 4% LDAO, 10-2 M o-phenanthroline, A8W1 cm = 0.2, resulted in less than 0.05 UQ/RC. The binding of ubiquinone to these reaction centers is shown in Fig. 2.
of different amounts of LDAO (1-4%) and o-phenanthroline (1-10 mM). All buffers were 10 mM Tris-HCl, pH 8.0. The RC's were then adsorbed onto a column of DEAEcellulose (Whatman DE 52), washed with 0.1% LDAO, eluted with 1 M NaCl (0.1% LDAO), and dialyzed against 0.025% LDAO. A large-scale preparative procedure for maximal removal of UQ was to adsorb RC's onto a column of DEAE-cellulose that was washed for 10 hr at 250C with 4% LDAO, 10 mM o-phenanthroline (1 liter/30 mg of RC's). The residual UQ in the RC's was extracted for 30 min at 350C in acetone-methanol (1:1, v:v), then purified, following the procedure of K. and A. Takamiya (22), and assayed by measuring the change in absorbance at 275 nm after reduction with NaBH4 [Ae275M = 12.25 X 103 Mcm'1] (23). Reconstitution of Reaction Centers. Reconstitution of UQ-depleted RC's was accomplished by two different methods. 14C-Labeled UQ-50 (Qlo) (about 5 X 10-5 M), solubilized in 1% LDAO, was added to RC's (As'l cm = 2, 1% LDAO) and incubated at 4°C for 10 hr. The RC's were separated from the unbound UQ by DEAE chromatography, as before, and their UQ content was assayed by liquid scintillation counting. An alternate method was used for other quinones that were expected to bind more weakly. Different ratios of UQ/RC (1, 5, 20), solubilized in 1% LDAO, were added to RC's (A 8'l cm = 20, 1% LDAO), incubated first at 25°C for 1 hr and then at 40C for 10 hr. EPR and optical samples were prepared by adding an equal volume of glycerol to the reconstituted RC's and freezing them in liquid nitrogen. This method was tested also with UQ-50j and found to give the same results as the first method. The iron content was measured by atomic absorption spectroscopy (Varian-Techtron model AA-5). Source of Quinones. The radiolabeled UQ was prepared by growing R. spheroides R-26 in the presence of [14C]acetate (0.3 mCi/liter). The [14C]UQ was extracted as described
ence
above and purified by thin-layer chromatography on silica gel sheets (Eastman), developed with benzene, eluted with ethanol, and crystallized from ethanol. The specific activity was 0.1 ;tCi/gnmol. This quinone has been previously shown to be UQ-50 (16). UQ-50, UQ-30, menadione, and phylloquinone were obtained from Sigma Chemical Co.; duroquinone and anthraquinone from Aldrich Chemical Co.; and plastoquinone-C from W. Butler (University of California at San Diego). Determination of Photochemical Activity. The photochemical activity at cryogenic temperatures was determined by two methods: (1) Optically by using the IR-2 mode of the Cary 14 as the strong (actinic) light source and measuring the bleaching at 890 nm. The sample was in thermal contact with a copper "cold finger" that was immersed in liquid nitrogen, and was in a Pyrex dewar with optically flat windows. (2) By measuring the amplitude of the light-induced EPR signal at g = 2.0026. The percent of activity agreed for both cases with the amplitude extrapolated to infinite light intensity obtained from a plot of reciprocal amplitude versus reciprocal light intensity. The kinetics of the light-induced changes were measured as described earlier (17). The EPR spectrum of the broad signal was obtained with light modulation (7, 17) (rather than magnetic field modulation). In this mode of operation the output signal is proportional to the absorption X" rather than the usually observed derivative
dX"/dH. EXPERIMENTAL RESULTS Removal of ubiquinone The UQ content of purified RC's was found to be close to 2 molecules/RC. UQ was removed by incubating RC's in the presence of LDAO and o-phenanthroline. By varying the concentration of protein, LDAO, o-phenanthroline, and the incubation period, different amounts of bound UQ were removed. The number of UQ's that remained bound to RC's under different conditions is shown in Fig. 1. It is seen that one UQ molecule is easily removed (Fig. 1, top two curves) while the removal of the second molecule requires more rigorous conditions (Fig. 1, bottom curve). RC's extracted twice under the most severe conditions (4% LDAO, 10-2 M ophenanthroline, A800' cm = 0.2) were labeled UQ-depleted; they contained about 0.05 UQ/RC and 1.05 i 0.05 Fe/RC. Reducing conditions were found to facilitate the removal of UQ; RC's incubated in the presence of 5 mM NaBH4 (0.5% LDAO, 50 mM Tris, pH 8) were found to lose all UQ in excess of 1.0 UQ/RC. Reconstitution of reaction centers with ubiquinone UQ-depleted RC's were reconstituted with various amounts of UQ. In order to improve the accuracy and ease of determination of UQ incorporation, radiolabeled ['4C]UQ was used. Fig. 2 shows the amount of UQ bound as a function of UQ added. The photochemical activity of untreated, UQ-depleted, and reconstituted RC's was measured at cryogenic temperatures. Two independent criteria were used for measuring the activity: (1) The light-induced change in absorbance of the 890 nm peak (Fig. 3a-c) and (2) the amplitudes of the two light-induced EPR signals (at g = 2.0026 and g = 1.8) corresponding to the donor (D+) and acceptor (A-) (Fig. 3d-f). In UQ-depleted RC's, the light-induced optical and EPR signals were virtually eliminated. However, in the absence of UQ a triplet EPR signal, as reported by Dutton, et al. (25), was observed (see insert in Fig. 3e). Upon addition of UQ to
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Proc. Nat. Acad. Sci. USA 72 (1975)
this low (2.1OK) temperature, the measurements were performed under partial microwave saturation of the narrow EPR line (16, 17). The quantitative relation between low-temperature photochemical activity and UQ content was determined on two sets of samples. In one, UQ was removed to varying degrees and in the other different amounts of UQ were added to UQ-depleted RC's. The results on both sets of samples are shown in Fig. 4. The light-induced optical changes and EPR amplitudes were normalized to untreated RC's. The dotted line, which approximates the experimental points fairly well, represents the expected light-induced changes if one UQ per RC is required for activity.
2.0
O05 1.0
3493
3
00
Reconstitution of reaction centers with other quinones In order to understand the structural requirements of the primary acceptor, other quinones were used to reconstitute UQ-depleted RC's. A list of quinones that were successfully incorporated is shown in Table 1. No detailed studies of incorporation, like those shown in Fig. 2, were performed; in most cases, however, it was found that a 5-times excess of added quinone resulted in close to maximum low-temperature activity. Two parameters that are believed to reflect the environment of the acceptor were measured: (1) The low-temperature decay time -TD (see previous section), and (2) the line width AH (full width at half amplitude) of the broad acceptor signal at g = 1.8. Both of these parameters changed when RC's were reconstituted with quinones other than UQ and are summarized in Table 1. In several cases, multiphasic decay kinetics were observed; the values of rD listed in Table 1 represent the initial decay.
-UQ ADDED [MOLE FRACTION]
FIG. 2. The binding of ubiquinone (UQ-50) to UQ-depleted reaction centers of R. spheroides R-26 (see Fig. 1). Reaction centers (A'ool cm = 2) in 1% LDAO were incubated with 14C-labeled ubiquinone for 10 hr at 41C.
the UQ-depleted RC's, both the light-induced optical and EPR changes were fully restored (compare Fig. 3a with Sc, and 3d with Sf). The low-temperature decay constant TD, obtained from the time dependence of the EPR amplitude, defined by A(t) = Ao e1t/TD was measured for untreated and reconstituted RC's. At 800K, TiD of the narrow EPR signal was found to be 28 4 2 msec for the untreated and 29 i 2 msec for the reconstituted RC's. The same values of TTD were obtained at 2.10K for both the broad (g = 1.8) and the narrow (g = 2.0026) EPR signals. In order to prevent the electron spinlattice relaxation from becoming the rate-limiting process at 0.8 b :zi
L'i
C-) .Z -cl m
0.6
CD U')
(0.4-
m -el
-i -CZ
(0.2
.2 Q-
C)
f 700
UNTREATED REACTION CENTERS d
750
900 800 850 -'WAVELENGTH [nml
950
UBIQUINONE ADDED BACK
UBIQUINONE REMOVED
T 2.I'K =
e-904 GHz
x'
-j
cr
t g2.0026 0
2
3
g=I.8 4
5
6
7
8
0
2
3
4
5
6
7
8
2
3
4
5
6
7
8
-'-MAGNETIC FIELD (kGoussJ FIG. 3. Low-temperature light-induced optical changes (upper) and EPR signals (lower) of reaction centers of R. spheroides R-26 with and without ubiquinone (UQ-50). The optical bleaching was accomplished by using the Cary in the IR-2 mode. The EPR signals were obtained with 4 Hz light modulation and are, therefore, proportional to the absorption x' and not to the usually observed derivative d x/dH. Microwave power 10-4 W, cavity Q0o 104. All samples were in 50% glycerol solution (optical path length of 1 mm, volume 0.1 ml). EPR samples had A800' cm = 15. Untreated reaction centers (d) and those to which ubiquinone was added back after extraction (f) show a narrow g = 2.0026 signal due to the donor (D+) and a broad g = 1.8 signal due to the acceptor (A-). Reaction centers from which virtually all the ubiquinone had been extracted show negligible bleaching (b) and a small EPR signal (e) attributed to the bacteriochlorophyll (BChl) triplet. The insert in (e) was obtained by increasing the light modulation frequency to 40 Hz (thereby eliminating the residual D+ signal), expanding the magnetic field scale and increasing the gain 5-fold.
Biophysics: Okamura et al.
3494
Proc. Nat. Acad. Sci. USA 72 (1975) Table 1. Low-temperature decay kinetics and EPR line widths of the acceptor in reaction centers of R. spheroides R-26 reconstituted with different quinones*
OPTICAL BLEACHING AA/A AT 890 nm + EPR AMPLITUDE (g92.0026) -____ EXPECTED IF ONE UQ/RC IS REQUIRED FOR ACTIVITY o
T = 80'K "
=
z
100
+>---o+0
4
+
*+
.,
80
UBIQUINONE-50
\0
8 ~\
_
60
LINE WIDTH
29±2
600±30
29±2
600± 30
32±2
680 ± 40
105 ±10
1000±60
37±3
740±40
34±2
760±40
135+10
740±40
28±2
600±30
_
X
+
DECAY TIME
Tt G~s tD msed]~ AH (GAUSS]*
STRUCTURE
COMPOUND
0+
y3 CH30
cn -:I M
40
C2:
20
C=
2
UBIQUINONE-30
2.
.0
b
PLASTOOUINONE-C
1.0 0.5 1.5 UO/RC (REMAINING AFTER EXTRACTION) o + ____
~36
OPTICAL BLEACHING AA/A AT 890nm EPR AMPLITUDE (9-2.0026) EXPECTED IF ONE UO/RC IS REQUIRED FOR ACTIVITY _
-o_
_
DUROQUINONE
CH3wCH3
MENADIONE
XCH3
0
T 80'K _+
CH3X
PHYLLOQUINONE
H
16 33
(VITAMIN K )
_
/
9
O/
ANTHRAQUINONE
T
IO
60
1.40
+,
UNTREATED REACTION CENTERS
2C
0
1.0 1.5 0.5 -UQ /RC (BOUND AFTER RECONSTITUTION)
2.0
FIG. 4. The relation between low-temperature photoactivity (normalized to untreated reaction centers) and the number of ubiquinones per reaction center. (a) obtained with reaction centers exposed to varying concentrations of LDAO and o-phenanthroline; (b) [14C]ubiquinone was added in varying amounts to reaction centers that had less than 0.05 ubiquinone per reaction center. The iron content (about 1 Fe/RC) remained unaffected by the extraction procedure.
DISCUSSION AND CONCLUSION We have shown that RC's bind two molecules of UQ which are distinguishable by their ease of removal with LDAO and o-phenanthroline (Fig. 1). The more loosely bound UQ is not required for the primary photochemistry and probably represents the secondary electron acceptor (6, 26). The ease of displacement of this UQ by o-phenanthroline offers a simple explanation of the inhibitory effect of o-phenanthroline on electron transfer to secondary electron acceptors (27). A possible mechanism of the removal of the UQ's by o-phenanthroline is the latter's binding to Fe2+, thereby displacing the UQ's. This suggests that both UQs' are situated in close proximity of the iron. The observation that reducing conditions facilitate the removal of one UQ is consistent with a mechanism of electron transfer in intact membranes that involves the release of the reduced secondary UQ from the RC to a UQ pool. The tightly bound UQ was found to be quantitatively correlated with the disappearance and recovery of the lowtemperature photochemical activity (Figs. 3 and 4). We conclude, therefore, that in R. spheroides it plays an obligatory role in the primary photochemistry. Several authors working with Rhodospirillum rubrum and Chromatium have concluded that UQ cannot be important in the primary photochemical act (28-30). These conclusions were based on the
* UQ-depleted reaction centers were incubated for 10 hr at 4'C in the presence of different quinones. For details, see Materials and
Methods.
t TD is the time required for the narrow EPR signal (g = 2.0026) to reach l/e of its amplitude after cessation of illumination (measured at T = 80WK). t AH is the full width at half amplitude of the broad g = 1.8 signal (see Fig. 3f), measured at 2.1'K.
presence of the primary reaction after extraction of UQ. It seems to us unlikely that different bacterial species should have developed totally different acceptorst, although this possibility cannot presently be excluded. Two possible alternative explanations for these conflicting results are that either the extraction conditions were not adequate to remove the tightly bound UQ, or that the photochemical activity at room temperature was mediated by an exogenous (diffusible) acceptor. The latter case points out the advantage of using low-temperature kinetic data as a criterion for "native" photochemical activity. The low-temperature decay time rD of the EPR signals, as well as the line width AH of the broad acceptor signal (g = 1.8), were found within experimental error to be the same for untreated and reconstituted RC's. Since these parameters are believed to be sensitive to the detailed environment of the acceptor (e.g., donor-acceptor distance), we conclude that the extraction of UQ and the reconstitution of the RC's were accomplished without altering the conformation of the active site. RC's reconstituted with other quinones showed deviations in the values of TiD and AH (Table 1). A tunneling mechanism for the decay, characterized by TiD, had been previously postulated (17). Consequently, TD should depend in a sensitive way on the shape of the potential barrier and the ionization (redox) potentials of the primary reactants (17). It will be instructive to test the tunneling hypothesis by makt
However, UQ.
as
shown in Table 1, different quinones might replace
Biophysics: Okamura et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
ing a detailed comparison of the observed TrD's and the respective redox potentials with the recently developed tunneling theory of Hopfieldt (31). Irrespective, however, of the detailed mechanism of charge recombination, the fact that replacing UQ with other quinones changes TD, whereas replacing iron by manganese does not, provides an additional argument in favor of assigning to UQ the major role in the primary photoact. It is noteworthy that in spite of the changes in TD and AH, photochemical activity was restored in RC's reconstituted with quinones of different structures. Although the structural requirements for reconstitution have not yet been fully explored, the present results indicate that neither the isoprenoid chain nor the methoxy groups of UQ are absolutely required for binding or photochemical activity. The observation of multiphasic kinetics indicates that in some of the reconstituted RC's the quinone did not assume a unique position. Having established that UQ plays an obligatory role in the primary photochemistry, what then is the function of iron? The chemical evidence [i.e., iron removal (10) and its replacement with manganese (16), the unchanged valence of iron upon reduction of the acceptor (18)] indicates that iron plays a relatively minor role in the primary photoact. On the other hand, spectroscopic evidence (i.e., the appearance of the g = 1.8 EPR signal) indicates that the presence of iron has a large influence on the EPR spectrum of the primary acceptor. These two sets of observations can be reconciled by postulating an Fe-UQ (ferroquinone) complex (11, 12). In the reduced form of the complex, the extra (unpaired) electron is mainly localized on the UQ; it can, however, interact magnetically with the nearby Fe2+. This interaction shifts and broadens (by two orders of magnitude) the quinone EPR resonance line. Preliminary calculations (H. Shore, personal communication) have shown that a magnetic exchange interaction (of the form JSI-S2), between a UQ radical (S, = %) and Fe2+ (S2 = 2) can give rise to g-values similar to those observed§. Since the energies involved in chemical reaction are much larger than magnetic energies, the fact that the EPR spectrum of the acceptor is greatly altered by the presence of iron does not mean that iron is essential for the primary photochemistry. Indeed, the indication that both UQ's are in close proximity to the iron leads to the hypothesis that the function of Fe is to serve as a path for electron transfer between the primary and secondary UQ's. We thank H. Shore for illuminating discussions and calculations on the magnetic properties of the iron-ubiquinone complex, L. Ackerson and S. Giovannoni for technical assistance, and W. Butler for the sample of plastoquinone. This work was supported by National Science Foundation Grant BMS-74-21413 and National Insti-
The
simplified tunneling
model of ref.
17
predicts
a
fourfold
change in 7rD for a 10% (about 0.1 eV) change in the redox potential of the acceptor. § EPR experiments model iron-quinone complexes such a~sFebenzoquinone have been performed by Blumberg and Peisach (32). on
3495
tutes of Health Grant USPHS GM-13191, and by a Career Development Award to M.Y.O. (1 K04 GM00106-01). 1. Norris, J. R., Uphaus, R. A., Crespi, H. L. & Katz, J. J. (1971) Proc. Nat. Acad. Sci. USA 68,625-629. 2. Feher, G., Hoff, A. J., Isaacson, R. A. & McElroy, J. D. (1973) Biophys. Soc. Abstr. 13, 61a (abstr. no. WPM-H7). 3. Norris, J. R., Druyan, M. E. & Katz, J. J. (1973) J. Am. Chem. Soc. 95,1680-1684. 4. Feher, G., Hoff, A. J., Isaacson, R. A. & Ackerson, L. C. (1975) Ann. N.Y. Acad. Sci. 244,239-259. 5. Norris, J. R., Sheer, H. & Katz, J. J. (1975) Ann. N.Y. Acad. Sci. 244, 261-280. 6. Parson, W. W. & Cogdell, R. J. (1975) Biochim. Biophys. Acta 416, 105-149. 7. Feher, G. (1971) Photochem. Photobiol. 14,373-88. 8. McElroy, J. D., Feher, G. & Mauzerall, D. (1970) Biophys. Soc. Abstr. 10, 204a. (abstr. no. FAM-E7) 9. Dutton, P. L., Leigh, J. S. & Reed, D. W. (1973) Biochim. Biophys. Acta 292, 654-664. 10. Loach, P. A. & Hall, R. L. (1972) Proc. Nat. Acad. Sci. USA 69,786-790. 11. Feher, G., Okamura, M. Y. & McElroy, J. D. (1972) Biochim. Biophys. Acta 267,222-226. 12. Bolton, J. R. & Cost, K. (1973) Photochem. Photobhol. 18, 417-421. 13. Clayton, R. K. & Straley, S. C. (1972) Biophys. J. 12, 12211234. 14. Slooten, L. (1972) Biochim. Biophys. Acta 275,208-218. 15. Bensasson, R. & Land, E. J. (1973) Biochim. Biophys. Acta 325, 175-181. 16. Feher, G., Isaacson, R. A., McElroy, J. D., Ackerson, L. C. & Okamura, M. Y. (1974) Biochim. Blophys. Acta 368, 135-139. 17. McElroy, J. D., Feher, G. & Mauzerall, D. C. (1974) Biochim. Blophys. Acta 333,261-278. 18. Debrunner, P. G., Schultz, C. E., Feher, G. & Okamura, M. Y. (1975) Biophys. J. 15, 226a (Abstract no. Th-PM-L12). 19. Cogdell, R. J. Brune, D. C. & Clayton, R. K. (1974) FEBS Lett. 45,344-347. 20. Okamura, M. Y., Steiner, L. A. & Feher, G. (1974) Biochemistry 13, 1394-1403. 21. Straley, S. C., Parson, W. W., Mauzerall, D. & Clayton, R. K. (1973) Blochim. Biophys. Acta 305,597-609. 22. Takamiya, K. & Takamiya, A. (1969) Plant Cell Physiol. 10,
363-373. 23. Pumphrey, A. M. & Redfearn, E. R. (1960) Biochem. J. 76, 61-64. 24. Feher, G., Isaacson, R. A. & McElroy, J. D. (1969) Rev. Sci. Instrum. 40, 1640-1641. 25. Dutton, P. L., Leigh, J. S. & Seibert, M. (1972) Biochem. Biophys. Res. Commun. 46,406-413. 26. Halsey, Y. D. & Parson, W. W. (1974) Biochim. Biophys. Acta
347,404-416. 27. Parson, W. W. & Case, G. D. (1970) Biochim. Biophys. Acta
205,232-245. 28. Noel, H., Van der Rest, M. & Gingras, G. (1972) Biochim. Biophys. Acta 275,219-230. 29. Ke, B., Garcia, A. F. & Vernon, L. P. (1973) Biochim. Biophys. Acta 292,226-236. 30. Bearden, A., Malkin, R., Parson, W. W. & Capolell, R. J., quoted on p. 138 of ref. 6. 31. Hopfield, J. J. (1974) Proc. Nat. Acad. Sci. USA 71, 36403644. 32. Blumberg, W. E. & Peisach, J. (1965) in Non-Heme Iron Proteins, ed. San Pietro, A. (The Antioch Press, Yellow Springs, Ohio), p. 101.
